Some soluble cellular enzymes in their natural environment have kinetic properties that are not evident under the conditions used in most enzymological studies. There is evidence that some soluble pathway-related enzymes are organized in cells, some associated with membranes, such that the pathway intermediates are channeled. These are important features of cells. The long term objective of this work is to obtain comprehensive information on the properties and organization of soluble enzymes in situ; this would extend our knowledge and understanding of normal cell structure and function and of regulatory mechanisms, as well as of the deviations from these which occur in disease. Several pathway-specific intermediates of urea synthesis are channeled, indicating that the enzymes of the pathway are organized in situ. The cytoplasmic urea cycle enzymes in permeabilized hepatocytes will be characterized with regard to their requirements for aspartate, ornithine, and ATP. Experiments involving inhibition of specific transporters and competition between endogenous and exogenous substrates will be done to obtain information on the channelling of intermediates. Immunohistochemical methods will be used to locate the cytoplasmic urea cycle enzymes in intact and permeabilized hepatocytes, and possibly in Bri j-treated and "nude" hepatocytes. Preparations of inner and outer mitochondrial membranes and of contact sites will be used to determine whether ornithine transcarbamylase, the ornithine transporter, and the arginase of the outer membrane form a complex; evidence will be obtained from sedimentation analysis, immunoblotting, and patterns of cross-linking.